Re: [ccp4bb] How to apply correction factors for translational NCS in PHASER?
Hi, If there is more than one strong unique Patterson peak, then Phaser currently doesn't know how to interpret the tNCS. If all the peaks can be interpreted as multiples of one one vector (taking symmetry into account) then you can use the NMOL argument of the tNCS command. Otherwise, you can get Phaser to only take account of the top peak by setting the minimum peak height. If you post or email the list of peaks, I might be able to give some specific advice (although I'll be on my way to the Cold Spring Harbor school most of the day). Best wishes, Randy Read Randy J. Read On 22 Oct 2013, at 23:35, Niu Tou niutou2...@gmail.com wrote: Dear All, I have a MR case which may contain two different molecules, ideally 1:1 ratio. Solvent content analysis indicates most likely to be 4~6 hetero molecules. The data shows at least 5 strong translational NCS vectors. When I tried to run PHASER, it always said correction factors NOT applied, but I have chosen to count it in the interface. Is there anyway I could use these NCS information, either by checking some options in the GUI of modifying the script? Many thanks! Best, Niu
[ccp4bb] stereo monitors/MAC
Hi, Having now had a chance (finally) to play with the Asus VG27A, I can say that (a) it works really well in stereo with both Coot and PyMol and (b) it is easier and nicer to use than the Zalmans. Though the technology is essentially the same (halving the vertical resolution), they have done two things better than the Zalmans: 1) - they don't just eliminate Pixels in the non-stereo sections of the screen like the menus. The menus become less easy to read (and completely weird in mono) but they can be read in stereo. This is a GOOD THING. 2) - there is a handy-dandy button at the bottom of the display that switches the screen into stereo from mono, and works fine in both coot and pymol. Heartily recommended for the diehards who like having the z-coordinates of their models correct. adrian
[ccp4bb] Structural Biology Faculty Position at Purdue University
* * Faculty Position in Structural Biology Purdue University The Department of Biological Sciences and the Markey Center for Structural Biology at Purdue University invite applications for a tenure-track faculty position in Structural Biology. We expect to fill this academic-year appointment at the Assistant Professor level, but appointment at the Associate level will also be considered. Applicants must have a Ph.D. or equivalent in biology, chemistry, physics or related field and at least 2 years of postdoctoral experience in X-ray crystallography, cryo-EM, NMR or other relevant field. The successful applicant will be required to establish a strong and independent research program in structural biology and to teach at the undergraduate and graduate levels. Applicants with research interests in cancer biology, drug discovery, energy signal transduction, infectious disease, large macromolecular complexes, membrane-based transport, molecular pathogenesis, neurobiology, viruses or viral-host interactions are encouraged to apply. The Markey Center for Structural Biology at Purdue is recognized worldwide for its leadership in structural biology of viruses, membrane proteins, receptors, signaling proteins and enzymes in addition to methods development in crystallography, NMR and electron microscopy. The Department has over 50 faculty members conducting research in a wide range of fields including those areas listed above, bioinformatics, computational chemistry and biology, ecology, evolutionary biology and vision research. The successful candidate will have opportunities to interact with colleagues in many departments and colleges including Chemistry, Medicinal Chemistry and Molecular Pharmacology, Computer Science and Physics, and to join centers such as the Center for Cancer Research and the new Center for Drug Discovery. The successful candidate will also have laboratory space in the newly constructed Hockmeyer Hall of Structural Biology and will have access to state-of-the-art shared resources across Purdue including a Titan Krios cryo-TEM, Bruker Avance-III 800 MHz NMR, Rigaku X-ray generators and detectors, and other advanced biophysical instrumentation available at the Bindley Bioscience Center and the Birck Nanotechnology Center. Applications must be submitted electronically to *sea...@bio.purdue.edu* as a PDF file that includes a detailed curriculum vitae, names and addresses of three referees, a 2 - 3 page summary of research interests, and a one-page statement on a vision for teaching. Inquiries should be directed to *Structural Biology Search Committee, Department of Biological Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907-2054*. Review of applications will begin *November 1, 2013 *and continue until the position is filled. Further information about the Department is available at http://www.bio.purdue.edu/. * * *Purdue University in an Equal Opportunity/Equal Access/Affirmative Action employer fully committed to achieving a diverse workforce.*
[ccp4bb] stereo monitors/MAC (try 3!)
Hi, (and apologies if ANYONE has seen this twice - CCP4BB rejected my first post as coming from an unknown (i.e. Leeds) address, and the second one because I had already posted from my Leeds address….) Here goes: Having now had a chance (finally) to play with the Asus VG27A, I can say that (a) it works really well in stereo with both Coot and PyMol and (b) it is easier and nicer to use than the Zalmans. Though the technology is essentially the same (halving the vertical resolution), they have done two things better than the Zalmans: 1) - they don't just eliminate Pixels in the non-stereo sections of the screen like the menus. The menus become less easy to read (and completely weird in mono) but they can be read in stereo. This is a GOOD THING. 2) - there is a handy-dandy button at the bottom of the display that switches the screen into stereo from mono, and works fine in both coot and pymol. Heartily recommended for the diehards who like having the z-coordinates of their models correct. adrian
Re: [ccp4bb] membrane protein optimization
Dear Frank, It is difficult to say something without the diffraction images but probably they are detergent ( or lipid) crystals. These crystals are in general soft and don't diffract well. My suggestion is to know well what your crystals really are before any optimization. Best, Isabel Dr Isabel De Moraes, MRSC Membrane Protein Laboratory Diamond Light Source Ltd, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, OX11 ODE, UK On 23 Oct 2013, at 17:22, crystalboy wrote: Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank [cid:7839c9ff-b567-4b4b-b191-07dc6c335f34@fed.cclrc.ac.uk] -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] membrane protein optimization
The problem you're having is a very common one, unfortunately. There are about 10,000 things you can try to improve diffractiongenerally going from low to solvable resolution is very, very hard. I would say the two most important ones are (i) to vary the detergent and (ii) to go to another homolog if (i) doesn't work. You don't mention which detergent you're using (DDM?), but the best chance of success would be to try harsher detergents, ie those that form small micelles (shorter chain maltosides, b-OG, LDAO etc). Give bicelles a try too, and LCP if you know someone with an LCP robot. If your protein is stable only in DDM you're out of luck and the best thing would be to look for an ortholog that is more stable. Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy [yyb...@gmail.com] Sent: Wednesday, October 23, 2013 5:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] membrane protein optimization Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank
Re: [ccp4bb] membrane protein optimization
Hi Frank, unfortunately it is very common with membrane protein crystals to get stuck with diffraction quality around 20A. From what you describe you could consider the following: a) revise the detergent you solubilize your MP with (e.g. use OG instead of DDM), and consider to change to another (shorter) detergent that might allow tighter crystal packing b) engineer your MP,mutate charged residues to alanine (check function!) c) try homolog protein d) try to crystallize in lipid cubic phase Best of luck! Susanne On Oct 23, 2013, at 9:22 AM, crystalboy wrote: Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank 20130917020056875.jpg Dr. Susanne Ressl Otto Hahn Fellow of the Max-Planck Society Stanford University School of Medicine James H. Clark Center 318 Campus Drive, Room E300 Stanford, CA 94305-5432 Phone: +1-650-736-1715
Re: [ccp4bb] membrane protein optimization
Hi Frank, Do not forget that membrane protein crystals are often fragile and difficult to manipulate. Finding good cryo condition can be difficult and small temperature variation can destroy crystals within minutes, this makes room temperature diffraction tests not always obvious. The time of harvest may also be critical. In addition to the previous suggestions, it is often usefull to check if there is a large amount of lipids in the sample. This can often easily be done by thin layer chromatography. HTH Daniel Le 23/10/2013 18:22, crystalboy a écrit : Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank
Re: [ccp4bb] Error with iMosflm 1.0.7 of CCP4 6.4.0 kit
Hi David
Nothing to do with the iMosflm developers. CCP4 has made some changes
that have caused this error to arise in 6.4.0 on Linux (this is not
to say that the iMosflm code didn't have the bug, just that it
doesn't appear with either our own version or with the same version
distributed with ccp4 6.3.0).
The guys at ccp4 have produced a fix which will be released shortly,
but they tell me this works;
For now a quick workaround is to add 2@1 after word exit in line
847 in ccp4-6.4.0/share/ccp4i/imosflm/src/controller.tcl.
--- src/controller.tcl 2012-11-30 20:46:13 +
+++ src/controller.tcl 2013-10-19 08:52:29 +
@@ -844,7 +844,7 @@
# If an executable has been named...
if {$l_executable != } {
# test the executable, by running it
- if {![catch {exec $l_executable exit} l_result]} {
+ if {![catch {exec $l_executable exit 2@1 }
l_result]} {
if { [regexp -nocase windows $::tcl_platform(os)] } {
set summary_filename [file join $::env(MOSDIR)
SUMMARY]
#set summary_filename [list $summary_filename]
Of course, my favourite fix is to run the version from our website
which does not show this bug.
Best wishes
On 23 Oct 2013, at Wed23 Oct 17:39, David Schuller wrote:
iMosflm developers:
I have installed CCP4 6.4.0, which includes iMosflm 1.0.7 and
ipmosflm 7.0.9.
This is on Scientific Linux 6.x, 64 bit distribution.
When I run iMosflm either from the command shell or from the ccp4i
interface, I get an error just like this:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg27243.html
iMosflm claims it cannot run without mosflm 7.0.9, followed by
output indicating that mosflm 7.0.9 runs. (See attached screen
capture of error message)
Note the linked description is for the previous versions of imosflm
amd mosflm, so this error seems to be a repeat.
I downloaded the iMosflm and ipmosflm executables from the iMosflm
web site, put ipmosflm in my executable directory, and this seems
to fix the problem.
You should get the CCP4 folks to investigate this and propagate a fix.
Cheers,
--
==
=
All Things Serve the Beam
==
=
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu
imosf.err.jpg
Harry
--
** note change of address **
Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick
Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH
Chairman of European Crystallographic Association SIG9
(Crystallographic Computing)
Re: [ccp4bb] membrane protein optimization
Hi Frank, The previous suggestions are great. In my case, I had soft crystals (bend from 180° to ~130-140°) in the loop using the LCP but they still diffracted. There are a lot of heroic stories on how some people solved a structure, you should just try as many ways as possible (do not only rely for a long time on only one condition or construct), and remember methods are just methods (i.e., there are hundreds of LCP robots in the world..). Protein engineering, homologs (maybe hyperthermophiles).. stabilization.. are great ways. Recently I had tested various crystals grown by vapor diffusion for a protein purified in different ways/detergents, so obtaining crystals is very common, but it's just the start. I also had crystals using bicelles but saw huge spots to 2 A (no patterns.. rather looked like detergent/lipid). Good luck toufic el arnaout From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy [yyb...@gmail.com] Sent: Wednesday, October 23, 2013 11:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] membrane protein optimization Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank
Re: [ccp4bb] membrane protein optimization
Dear Frank, Toufic is right. The trick is no trick, you just have to try everything. In my experience if u have a protein crystals in a mild detergent and it is very fragile no point going to a harsh or shorter chain detergent like OG. I got crystals in NM (9 C) and they were fragile and dissolved after 1 day. I could only get stable crystals when I moved to DM (10 C) and DDM (12 C). Once you get a stable crystal you may consider dehydrating them in higher PEG so as to improve protein protein contact. Again the trick is no trick, you just try everything suggested. Good luck with your trials. Emmanuel Nji. On 23 October 2013 19:46, El Arnaout, Toufic elarnao...@biochem.wustl.eduwrote: Hi Frank, The previous suggestions are great. In my case, I had soft crystals (bend from 180° to ~130-140°) in the loop using the LCP but they still diffracted. There are a lot of heroic stories on how some people solved a structure, you should just try as many ways as possible (do not only rely for a long time on only one condition or construct), and remember methods are just methods (i.e., there are hundreds of LCP robots in the world..). Protein engineering, homologs (maybe hyperthermophiles).. stabilization.. are great ways. Recently I had tested various crystals grown by vapor diffusion for a protein purified in different ways/detergents, so obtaining crystals is very common, but it's just the start. I also had crystals using bicelles but saw huge spots to 2 A (no patterns.. rather looked like detergent/lipid). Good luck toufic el arnaout From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy [yyb...@gmail.com] Sent: Wednesday, October 23, 2013 11:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] membrane protein optimization Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank -- Dr. Emmanuel Nji Department of Biochemistry and Biophysics Centre for Biomembrane Research Stockholm University 106 91 Stockholm Sweden Cell: 0046736457727
[ccp4bb] MacBook Pro graphics card options
Hi everyone, Sorry for being a bit off topic, but I thought this group would be great for advice. I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn as to whether I need integrated vs discrete graphics for structural biology, including molecular modelling, especially since the latest advances by Intel in terms of integrated graphics. Right now with the new releases, the options are between Intel Iris Pro (5200 series) and Intel Iris Pro + Nvidia GT 750M. Thoughts? Thanks everyone! Kristin - Kristin Low Ph.D. Candidate Queen's University Department of Biomedical and Molecular Sciences Botterell Hall, Room 645 Kingston, ON Canada K7L 3N6 tel: +1-613-533-3019 -
Re: [ccp4bb] MacBook Pro graphics card options - what about Mavericks?
Hi, Can I profit from Kristin's question and add one: is it too soon to know if the crystallography software (CCP4, Coot, Autobuster, XDS) will work fine with Mavericks (Mac OS X 10.9)? (I remember a bit of trouble when Lion came off). Carlos Em 23 oct. 2013, às 22:10, Kristin Low kristin@queensu.ca escreveu: Hi everyone, Sorry for being a bit off topic, but I thought this group would be great for advice. I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn as to whether I need integrated vs discrete graphics for structural biology, including molecular modelling, especially since the latest advances by Intel in terms of integrated graphics. Right now with the new releases, the options are between Intel Iris Pro (5200 series) and Intel Iris Pro + Nvidia GT 750M. Thoughts? Thanks everyone! Kristin - Kristin Low Ph.D. Candidate Queen's University Department of Biomedical and Molecular Sciences Botterell Hall, Room 645 Kingston, ON Canada K7L 3N6 tel: +1-613-533-3019 -
Re: [ccp4bb] MacBook Pro graphics card options - what about Mavericks?
I'm currently using CCP4/COOT (from the official installer) and autoPROC on Mavericks without any problems. I imagine the rest of the global phasing tools will work nicely. I had issues with fink, you have to use a branch from github as well as manually install and update the Command Line Tools just to get fink working... F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder On Oct 23, 2013, at 3:16 PM, Carlos Kikuti kik...@gmail.com wrote: Hi, Can I profit from Kristin's question and add one: is it too soon to know if the crystallography software (CCP4, Coot, Autobuster, XDS) will work fine with Mavericks (Mac OS X 10.9)? (I remember a bit of trouble when Lion came off). Carlos Em 23 oct. 2013, às 22:10, Kristin Low kristin@queensu.ca escreveu: Hi everyone, Sorry for being a bit off topic, but I thought this group would be great for advice. I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn as to whether I need integrated vs discrete graphics for structural biology, including molecular modelling, especially since the latest advances by Intel in terms of integrated graphics. Right now with the new releases, the options are between Intel Iris Pro (5200 series) and Intel Iris Pro + Nvidia GT 750M. Thoughts? Thanks everyone! Kristin - Kristin Low Ph.D. Candidate Queen's University Department of Biomedical and Molecular Sciences Botterell Hall, Room 645 Kingston, ON Canada K7L 3N6 tel: +1-613-533-3019 -
Re: [ccp4bb] Error with iMosflm 1.0.7 of CCP4 6.4.0 kit
Hi David, I've just renamed the ipmosflm in $CCP4/bin to ipmosflm_new and then defined MOSFLM_EXEC as /path/to/ccp4-6.3.0/bin/ipmosflm. CCP4 6.3 needs to be installed for this to work. Andreas On 23/10/2013 5:39, David Schuller wrote: iMosflm developers: I have installed CCP4 6.4.0, which includes iMosflm 1.0.7 and ipmosflm 7.0.9. This is on Scientific Linux 6.x, 64 bit distribution. When I run iMosflm either from the command shell or from the ccp4i interface, I get an error just like this: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg27243.html iMosflm claims it cannot run without mosflm 7.0.9, followed by output indicating that mosflm 7.0.9 runs. (See attached screen capture of error message) Note the linked description is for the previous versions of imosflm amd mosflm, so this error seems to be a repeat. I downloaded the iMosflm and ipmosflm executables from the iMosflm web site, put ipmosflm in my executable directory, and this seems to fix the problem. You should get the CCP4 folks to investigate this and propagate a fix. Cheers, -- Andreas Förster Crystallization and Xray Facility Manager Centre for Structural Biology Imperial College London
Re: [ccp4bb] MacBook Pro graphics card options
On Wed, Oct 23, 2013 at 1:10 PM, Kristin Low kristin@queensu.ca wrote: I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn as to whether I need integrated vs discrete graphics for structural biology, including molecular modelling, especially since the latest advances by Intel in terms of integrated graphics. Right now with the new releases, the options are between Intel Iris Pro (5200 series) and Intel Iris Pro + Nvidia GT 750M. It depends on how demanding your graphics needs are. I have no experience with the Iris Pro chips, but I've been using a MacBook Air from late 2011 almost exclusively for most of the last two years, including heavy use of Coot and PyMOL, and the only times I've been annoyed by slow graphics is when I'm trying to visualize a very large and/or high-resolution region of density. And even that doesn't happen too often. Most of the time it runs very smoothly. However, there are some persistent glitches with the graphical display in Coot - sometimes I get weird visual artifacts, or messed up depth perception. Whether this is the fault of Coot, XQuartz, or Intel is unknown. But speed is not an issue. The premium for the model with the Nvidia chip is quite steep at $600 (perhaps it's less with academic discount?). I don't think the graphics upgrade alone is worth it - but I'd be very tempted by the faster processor, doubled memory, and doubled SSD, all of which will come in handy when refining or rendering. Disclaimer: I know absolutely nothing about the availability of stereo options with any of these systems. It's possible the NVidia card has additional capabilities in that respect. -Nat
